LINE-1 DNA methylation levels in DPPA3 KO overexpressing DPPA3 mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina MiSeq
ORGANISM(S): Mus musculus
SUBMITTER: Sebastian Bultmann
PROVIDER: E-MTAB-9653 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Tet1, Tet2 and Tet1/Tet2 catalytic mutants as well as DPPA3 KO ESCs harboring a doxycyclin inducible Dppa3 transgene were induced for several days to monitor LINE-1 methylation levels.
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Project description:Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining pulse-chase experiments in engineered CRC cells and transcriptomic analyses, we identified DPPA3 as a master regulator of slow-cycling phenotype in CRC. We find that DPPA3 stabilizes HIF1a even in normoxia thus limiting nuclear CCNB1 levels and represses DNA replication and cell cycle programs resulting in a slow cell-cycle phenotype. Down-regulation of HIF1a partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of patient samples, we find that DPPA3 is a predictive biomarker of CRC chemotherapeutic resistance and tumor relapse. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1a axis to support tumor persistence under therapeutic stress and provides key insights on the molecular regulation of tumor cell slow-cycliness and chemoresistance. This dataset comprises DNA methylation data of SW1222 CRC cells subjected to DPPA3 overexpression for 5 days.
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Project description:DNA methylation profiling using RRBS in wild type and Dppa3 knockout ESCs.
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Project description:Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.
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Project description:Background: DNA methylation is important for maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2’-deoxycytidine (5-aza-2’-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation. Results: We found that inhibition of ribonucleotide reductase (RNR) induced expression of the reporter. RNR inhibition potentiated the effect of 5-aza-2’-dC by enhancing its DNA incorporation, thereby decreasing genome-wide DNA methylation levels. Since both 5-aza-2’-dC and RNR-inhibitors are used in the treatment of hematological malignancies, we treated myeloid leukemia cell lines with 5-aza-2’-dC and the RNR inhibitor hydroxyurea, and observed synergistic inhibition of cell growth and decreases in genome-wide DNA methylation. Conclusions: Taken together, our study identifies a drug combination that enhances DNA demethylation by altering nucleotide metabolism. We demonstrate that XCR assays can be used to optimize epigenetic activity of drug combinations. Reduced representation bisulfite sequencing (MspI,~40-220bp size fraction) of murine and human cells.
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Project description:Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies focused on epigenetic factors that affect ESC self-renewal and pluripotency. Through our laboratory's previous studies on ESCs pluripotency, we found that Dppa3, as a Naive state marker gene, is of great significance to the transformation of mESCs pluripotency. However, the influence of overexpression of Dppa3 in ESCs on mESCs status has not been determined. Our results show that overexpression of Dppa3 induces global DNA demethylation, which is beneficial to the maintenance of pluripotency, but its differentiation ability is significantly impaired. In mESCs, Dppa3 regulates mESCs pluripotency by inhibiting de novo methylation pathway, maintaining methylation pathway, promoting demethylation pathway Tet2, up-regulating active histone modification and down-regulating heterogeneous histone modification. The 2C-like state of ESCs recapitulates key aspects of the two-cell stage mouse embryo both phenotypically and molecular, which providing a cellular model to investigate the progress of ZGA. Our results found Dppa3 promote facilitate 2C-state conversion.
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Project description:We performed whole-genome bisulfite sequencing of Dppa3 knockout fully grown oocytes (FGOs).
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Project description:LINE-1 DNA methylation levels in DPPA3 KO overexpressing DPPA3 mutants
| PRJEB40756 | ENA
Project description:In this study, we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed whole-genome bisulfite sequencing (WGBS) experiments to compare the DNA methylation landscapes of conventional EpiSCs and rsEpiSCs. Compare the DNA methylation profiles in 2 pluripotent stem cell types (LP-EpiSCs and conventional EpiSCs) in mouse. Two replicates are examined for each cell type.
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Project description:Extracellular vesicles (EVs) released by cells contain mRNAs, miRNAs, lncRNAs, lipids, and proteins, playing crucial roles in cell-cell communication. While full-length mRNA transcripts have been documented in EVs secreted by cancer cells, there are no reports on full transcripts secreted by embryos. Our study aimed to identify extracellular vesicle mRNAs in the culture media of bovine embryos and investigate their roles in embryo-mother communication. Following the isolation of EVs from in-vitro fertilization media samples and RNA sequencing, we identified full mRNA transcripts of three genes: SLBP2, ACCSL, and DPPA3. We selected DPPA3 for further investigation. To examine the role of DPPA3 in embryo-mother communication, an in vitro transcribed mRNA of DPPA3 was transfected into bovine endometrial epithelial cells. Transfected and control cells were subsequently analyzed for RNA sequencing to assess the effects of DPPA3 on gene expression. A total of 24 genes were found to be upregulated, and one gene was downregulated (FDR < 0.01) following DPPA3 transfection. Among the 18 annotated genes, 17 have known functions in pregnancy recognition in ruminants or have been previously identified as upregulated in pregnant animals. In addition to RNA sequencing, transfected and control cells underwent proteomic analysis. A total of 34 proteins were differentially expressed (FDR < 0.01, p < 0.05, fold change > 1.5), with 19 upregulated and 15 downregulated. Two proteins, ISG15 and MX1, overlapped with the differentially expressed mRNAs. To mimic the natural transfer of EVs from embryos to endometrial cells, we performed co-culture of day 8 blastocysts or supplemented the cells with embryo-conditioned culture media. DPPA3 expression was detected in endometrial cells exposed to embryo-conditioned media after just 30 minutes, with slight upregulation of ISG15 and MX1 also observed. The expression of these classic interferon-stimulated genes in endometrial cells increased from 0.5 to 10 hours when exposed to blastocysts or embryo-conditioned media. Overall, these results demonstrate that DPPA3 mRNA secreted from embryos may play a role in early embryo-mother communication and maternal recognition of pregnancy prior to major interferon tau secretion. Importantly, our study highlights the significant role of EVs in cell-cell communication through mRNA signaling from the embryo to the mother.
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